It is proposed to develop methods for isolation, growth and unequivocal identification of strains of Moraxella, Legionella, Neisseria, and other gram-negative bacteria that reside in various locations in the human body, many such bacteria being particularly difficult to identify with certainty using conventional methods. Long range goals include the development of a general transformation assay procedure for identification of any bacterium of clinical significance. The ability to distinguish species of Moraxella and other gram-negative bacteria precisely will provide an opportunity to assess the possible role of these organisms in human disease. To accomplish these goals we will develop assays in which DNA from an unknown strain specifically transforms a discernible marker in a competent strain of the same organism, DNA from unrelated organisms being ineffective. A general transformation procedure for identification of strains of various bacteria will be devised making use of recombinant DNA methodology to incorporate markers of a species of interest into a plasmid, followed by introduction of the hybrid plasmid into a naturally competent host strain. Transformation of the marker in the hybrid plasmid that results in a detectible changes in a characteristic of the host bacterium should make it possible to identify specifically DNA orginating from strains of the species of interest. We plan to devise new plating media that will permit ready isolation of Legionella species as well as the establishment of transformation assays for their diagnosis. Similar studies, including physiological investigations, are planned for Moraxella nonliquefaciens and for Moraxella bovis, two bacterial species that have been shown to be involved in infections of the eye. Cysteine biosynthesis in Legionella and Neisseria will be studied in order to improve growth media for Legionella and to make possible a specific assay for Neisseria gonorrhoeae involving transformation of cysteine auxotrophs. Naturally occurring auxotrophic lesions found in most strains of N. gonorrhoeae will be transferred to a prototrophic strain to obtain a set of single marker strains that can be used in epidemiological studies as well as in investigations of the origin of these nutritional lesions.